Our determination of the structure of the E1E2 pyruvate dehydrogenase complex led to a number of important questions about the molecular mechanisms of active site coupling in these enzyme complexes. For example, would there be differences in the way E1 and E3 enzymes would be arranged in the outer shell? Are the linker regions structured or bundled together as they emerge from the core? Is the size of complex determined by protein-protein interactions at the outer shell, or by properties of the linker? What is the trajectory of the swinging arm of the enzyme that couples pyruvate decarboxylation to acetyl CoA synthesis? These and related questions constitute our ongoing studies of this complex. Using cryo-electron microscopy, we carried out a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100 kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of about 75 between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 co-ordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain. In a related set of studies we have addressed another important unresolved question, which is to determine whether the gap between the central E2 core and the outer enzyme shell is maintained by virtue of protein-protein interactions in the outer shell or by stiffness of the linker region connecting the core to the peripheral subunit binding domain. Using a combination of circular dichroism, analytical ultracentrifugation and solution NMR studies we have obtained evidence that the peptide corresponding to the linker region has an extended conformation with a persistence length of 75-89 , consistent with the observed size of the gap. Cryo electron tomography of individual complexes with varying occupancies of enzymes in the outer shell confirmed unequivocally that the annular between the core and the outer shell was maintained even at very low E1 or E3 occupancies. These studies demonstrate that the inner linker region of PDH enzymes are critical structural elements, serving to maintain the annular gap required for coupling the decarboxylation of pyruvate in the outer shell to the synthesis of acetyl CoA in the core.